Cell cycle-dependent gene networks for cell proliferation activated by nuclear CK2α complexes

Functional connection between activation of nuclear CK2α by phosphorylation which leads to association of CK2α with transcriptional start sites, and activation of histone genes, growth-related genes, and rDNA for cell proliferation.

We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original microscopy and blot data images before submitting your revision.***- --------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): General Comments: In this manuscript, the authors conduct a detailed examination of the activation and function of nuclear CK2 during cell proliferation.Multiple phosphorylation sites were examined, and the association of CK2alpha with transcriptional start sites was studied in detail.The work involves both proteomic and ChiP-seq approaches.The paper is very clearly written.It represents a major piece of work that advances the field in multiple aspects.
2. The data are strongly supportive of all of the conclusions that are drawn, with the caveat of the issue raised below concerning reproducibility.
3. There are several issues, most involving data presentation: a.The abstract requires English editing.In addition, an abstract typically includes more information.For example, in the sentencebeginning "This phosphorylation appears..." there is no indication of what types of experiments led to the conclusion; also the conclusion is rather weakly stated.b.There needs to be more attention to scientific reproducibility.The reviewer did not see any use of statistics in the paper.This reviewer is not a believer in excessive use of statistics, but many of the figures look odd without it (e.g., Figure 1F) as the addition of statistical information helps to direct the reader to the most important parts of each figure.The authors do not indicate how many replicate experiments were performed, or how many replicate measurements are reflected in each error bar.Overall, this is a very important issue that must be addressed.c.All of the labels that read "Blot with CK2alpha" should be "Blot for..." or better yet just "CK2alpha".They need to be consistent...Figure 3A is labelled "anti-CK2alpha".d.In Figure 6C, it is not apparent from the images what is being blotted for.
Reviewer #2 (Comments to the Authors (Required)): This manuscript describes the control of nuclear translocation of the protein kinase, CK2alpha by phosphorylation.Evidence is presented showing that (i) CK2a is phosphorylated at Ser7, S194 and S287; (ii) pSer7 appears necessary for nuclear translocation and serum stimulated cell proliferation; (iii) CK2 ChIP seq identifies promoter and genic loci for genes controlled in late G1 and ribosome biogenesis; (v) CK2a coimmunoprecipitates with regulators of late G1 and rRNA transcription, including RNAPII subunits, histones, and RNA helicases.The findings are taken to suggest that CK2 may facilitate G1 progression and ribosome biogenesis by associating with nuclear and nucleolar transcriptional regulators.Therefore, the study supports and expands new knowledge by identifying candidate targets for nuclear CK2a.
Strengths of the study are the numerous screens comparing WT, CK2-knock out, and CK2-S7A cells by transcriptome profiling, ChIP-seq, and co-immunoprecipitation.Experiments are described adequately and the data analysis appears sound.This is a rich dataset that will be useful to many researchers for understanding nuclear function(s) of CK2, which so far have been elusive.That many of these screens converge on genes involved in late G1 and rRNA transcription will be of particular interest to this field.Weaknesses are that the findings are mostly correlative.Conceivably, some of the transcriptional or co-IP responses that are altered in CK2-KO are secondary effects of the KO on cell cycle inhibition.Nevertheless, the data resource alone represent an impressive amount of work, and the co-IP data nicely ties the screening results together to support the hypothesis that CK2 functions through direct interactions with RNAPII and transcription regulators.Overall, the data generated by this paper should be valuable for understanding CK2 mechanisms and explaining the yet poorly understood requirement for this kinase in cell cycle progression.2. The data are strongly supportive of all of the conclusions that are drawn, with the caveat of the issue raised below concerning reproducibility.
We thank the Reviewer for his/her positive feedback on our manuscript.We have responded to all comments below.
3. There are several issues, most involving data presentation: a.The abstract requires English editing.In addition, an abstract typically includes more information.For example, in the sentence beginning "This phosphorylation appears..." there is no indication of what types of experiments led to the conclusion; also the conclusion is rather weakly stated.
We thank the reviewer for calling this to our attention.To address this comment, we have revised the Abstract.It now reads as follows.
Nuclear expression of protein kinase CK2α is reportedly elevated in human carcinomas, but mechanisms underlying its variable localization in cells are poorly understood.This study demonstrates a functional connection between nuclear CK2 and gene expression in relation to cell proliferation.Growth stimulation of quiescent fibroblasts and phospho-proteomic analysis identified a pool of CK2α that is highly phosphorylated at serine 7. Phosphorylated CK2α translocates into the nucleus, and this phosphorylation appears essential for nuclear localization and catalytic activity.Protein signatures associated with nuclear CK2 complexes reveal enrichment of apparently unique transcription factors and chromatin remodelers during progression through G 1 phase of the cell cycle.Chromatin immunoprecipitation-sequencing profiling demonstrated recruitment of CK2α to the active gene loci, more abundantly in late G 1 phase than in early G 1 , notably at transcriptional start sites of core histone genes, growth stimulus-associated genes, and ribosomal RNAs.Our findings reveal that nuclear CK2α complexes may be essential to facilitate progression of the cell cycle, by activating histone genes, triggering ribosome biogenesis, specified in association with nuclear and nucleolar transcriptional regulators.
b.There needs to be more attention to scientific reproducibility.The reviewer did not see any use of statistics in the paper.This reviewer is not a believer in excessive use of statistics, but many of the figures look odd without it (e.g., Figure 1F) as the addition of statistical information helps to direct the reader to the most important parts of each figure.The authors do not indicate how many replicate experiments were performed, or how many replicate measurements are reflected in each error bar.Overall, this is a very important issue that must be addressed.
We agree.To address this comment, we checked the original data and added statistical information to the figures.We also added numbers of replicate experiments and replicate measurements to the figure legends.Additionally, Figure EV2A has now been corrected, after re-calculating our ChIP-Seq data.This re-calculation did not affect our interpretation of the results.
Happily, it strengthened correlations between the two factors.It can be easily reproduced by the reader, using deepTools algorithms in standardization methods and procedures.c.All of the labels that read "Blot with CK2alpha" should be "Blot for..." or better yet just "CK2alpha".They need to be consistent...Figure 3A is labelled "anti-CK2alpha".
We thank the Reviewer for pointing this out.We have made the suggested alterations by changing the label "Blot with CK2α" to just "CK2α."Concerning Figure 3D, which demonstrates association of RNA polymerase II with CK2a, a blot for CK2a is now included in order to better support the conclusions.d.In Figure 6C, it is not apparent from the images what is being blotted for.We acknowledge that Figure 6C was poorly labeled.We have added the name of the target molecule ("Flag") to the blot in the figure .Reviewer #2 (Comments to the Authors (Required)): This manuscript describes the control of nuclear translocation of the protein kinase, CK2alpha by phosphorylation.Evidence is presented showing that (i) CK2a is phosphorylated at Ser7, S194 and S287; (ii) pSer7 appears necessary for nuclear translocation and serum stimulated cell proliferation; (iii) CK2 ChIP seq identifies promoter and genic loci for genes controlled in late G1 and ribosome biogenesis; (v) CK2a coimmunoprecipitates with regulators of late G1 and rRNA transcription, including RNAPII subunits, histones, and RNA helicases.The findings are taken to suggest that CK2 may facilitate G1 progression and ribosome biogenesis by associating with nuclear and nucleolar transcriptional regulators.Therefore, the study supports and expands new knowledge by identifying candidate targets for nuclear CK2a.
Strengths of the study are the numerous screens comparing WT, CK2-knock out, and CK2-S7A cells by transcriptome profiling, ChIP-seq, and co-immunoprecipitation.Experiments are described adequately and the data analysis appears sound.This is a rich dataset that will be useful to many researchers for understanding nuclear function(s) of CK2, which so far have been elusive.That many of these screens converge on genes involved in late G1 and rRNA transcription will be of particular interest to this field.Weaknesses are that the findings are mostly correlative.
Conceivably, some of the transcriptional or co-IP responses that are altered in CK2-KO are secondary effects of the KO on cell cycle inhibition.Nevertheless, the data resource alone represent an impressive amount of work, and the co-IP data nicely ties the screening results together to support the hypothesis that CK2 functions through direct interactions with RNAPII and transcription regulators.Overall, the data generated by this paper should be valuable for understanding CK2 mechanisms and explaining the yet poorly understood requirement for this kinase in cell cycle progression.
We thank the Reviewer for this positive comment on our manuscript.Concerning the comment, "Weaknesses are that the findings are mostly correlative.Conceivably, some of the transcriptional or co-IP responses that are altered in CK2-KO are secondary effects of the KO on cell cycle inhibition", we agree.We found that CK2 concentrates in proximity to promoters as the cell cycle progresses and actively participates in gene expression by interacting with transcription factors and Pol2.However, how CK2 cooperates in gene expression with RNA Polymerase II is uncertain.
We do not know whether CK2 directly binds to DNA like a transcription factor, activates RNA Polymerase II by phosphorylation, activates RNA Polymerase II by phosphorylating and removing regulatory factors, or phosphorylates histone proteins or chromatin remodelers to modify higher-order structure of chromatin, etc.Therefore, it will be important to clarify details of the molecular function of CK2 in proximity to promoters.We hope to publish the results as we proceed with verification.Fortunately, we have established unique, high-quality, CK2-specific antibodies, clones 6A and 16C, and analysis using them is expected to yield valuable insights.Thank you for submitting your revised manuscript entitled "Cell cycle-dependent gene networks for cell proliferation activated by nuclear CK2 complexes".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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1 Reviewer # 1 (
Comments to the Authors (Required)): General Comments: In this manuscript, the authors conduct a detailed examination of the activation and function of nuclear CK2 during cell proliferation.Multiple phosphorylation sites were examined, and the association of CK2alpha with transcriptional start sites was studied in detail.The work involves both proteomic and ChIP-seq approaches.The paper is very clearly written.It represents a major piece of work that advances the field in multiple aspects.
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